1. Field of the Invention
The invention is directed to methods and devices for high-resolution microscopic imaging of a sample labeled with a fluorescent dye, wherein the sample is illuminated sequentially in a plurality of phases by structured, pulsed excitation light, and the fluorescent light emitted by the sample is recorded for each phase in a respective structured individual image so that a result image with enhanced resolution can be reconstructed from the individual images.
2. Description of Related Art
Due to the fact that the light received from the sample is diffracted in the microscope objective, the resolving power of microscopes depends upon the aperture of the objective and the wavelength of the light. Since the usable wavelength range of visible light is finite, the resolving power of a microscope is fundamentally limited (Abbe, 1873). As it relates to the spatial frequencies of the sample which are to be imaged, this means that the support of the optical transfer function (OTF) of the microscope is limited in frequency space to a finite region around the coordinate origin. Consequently, the microscope can only image those spatial frequencies lying within the center interval in which the support does not vanish.
By means of a structured illumination of the sample (structured illumination microscopy (SIM)), the resolving power can be improved, laterally and axially, approximately by a factor of two when the excitation intensity of the illumination and the emission intensity of the sample are in a linear relationship with one another. SIM is disclosed in U.S. Pat. No. 5,671,085, for example. It is based on the generation of a spatial light structure on the sample to be analyzed, for example, by means of a sinusoidal interference of the illumination light behind an optical grating. Due to the convolution of the excitation structure with the point spread function of the microscope objective in the spatial domain, a region of spatial frequencies of the sample structure lying outside the support of the OTF in the frequency domain is shifted to the center support interval, where they overlap the original spatial frequency intensities in that region. The light structure is generated sequentially in a plurality of different phase positions, and an individual image is recorded in each phase position. With the aid of an appropriate equation system, a consistent result image can be reconstructed from the individual images containing the superimpositions of the shifted spatial frequencies and original spatial frequencies, which result image contains the original spatial frequencies of the support interval as well as the original, higher spatial frequencies that have been shifted into the support interval in the meantime by the structured illumination. Therefore, the result image has a higher resolution than a conventional single recording with uniform illumination. However, taking multiple images with different phase positions and orientation of the structuring requires a highly stable optical arrangement and sample throughout the entire measuring process. Further, the required multiple recording reduces the effective frame rate. Also, the structuring must be projected into the sample in a highly homogeneous manner (constant frequency and phase in the structuring).
A considerable improvement in resolving power can be achieved by exciting the sample (by illumination or in some other manner) in such a way that there exists a nonlinear relationship between the excitation intensity and the light intensity emitted by the sample (saturated pattern excitation microscopy (SPEM)). SPEM is disclosed, for example, in DE 199 08 883 A1, the disclosure of which is hereby incorporated in its entirety. In fluorescence microscopy, a nonlinear excitation is achieved, for example, by a high illumination intensity leading to a partial saturation of the excitation of the fluorescent dye in the area of the illumination structure. In this way, spatial frequencies of object structures even higher than those in SIM are shifted into the OTF support interval. By taking the nonlinear interaction into account in the equation system to be solved, these higher frequencies can also be reconstructed. Compared to SIM, however, smaller phase steps and, therefore, even more individual images are required in SPEM. The nonlinear sample interaction encumbers the sample and sample dyes by bleaching. In addition, the nonlinearity depends not only on the illumination conditions but also on the local environmental conditions in the sample. As a result, distinctly different nonlinearities may be achieved at different locations in the sample, which makes reconstruction of the result image more difficult. In some cases, the environmental conditions in the sample can be so unfavorable for a nonlinear interaction that the use of SPEM is impossible.
In SIM and SPEM, the resolution which can be achieved by reconstruction is fundamentally limited by the signal-to-noise ratio (SNR) in the recording of individual images.